Blood glucose level is an extremely crucial indicator in clinical diagnosis as an important marker for diabetes. In addition, in fermentative production using microorganisms, determination of glucose concentration is an important parameter for process monitoring. A glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme (PQQGDH) has a high oxidation activity for glucose and it does not require oxygen as an electron acceptor because PQQGDH is a coenzyme-linked enzyme. Thus PQQGDH is an promising enzyme to be applied to assay techniques, for example, as a sensing element of a glucose sensor.
In order to assay glucose by an enzyme electrode having PQQGDH immobilized on its surface, it is necessary to add an electron mediator to the assay system for transferring electrons from PQQ, which is a redox center of PQQGDH, to the electrode. Accordingly, there are disadvantages in that the performance of the electrode is limited depending on the stability or solubility of the electron mediator, and the background for measurement is increased by the reaction of a contaminant with the electron mediator. Furthermore, as the electron mediator is not suitable for use in vivo, the application of PQQGDH to a glucose sensor implanted in the body has been limited. In order to solve these problems, a method of immobilizing PQQGDH on an electrode with an electron transfer protein has been proposed (WO 02/073181). In this method, however, it is necessary to use extremely excess moles of electron transfer protein, which may lead to a cost problem. Thus, there has been a demand in the art for a sensing device of a “direct electron transfer-type” glucose sensor which requires no electron mediators.
Prior art documents related to the present invention includes: J. Okuda, J. Wakai, N. Yuhashi, K. Sode, Biosensors & Bioelectronics 18 (2003) 699-704; J. Okuda, J. Wakai, K. Sode, Anal. Lett. 35 (2002) 1465-1478.